The present invention relates in general to methotrexate derivatives and in particular to such derivatives which are incapable of forming polyglutamates thus having reduced cytotoxicity.
Methods are known for preparing methotrexate and similar compounds. See for example, U.S. Pat. Nos. 4,080,325 to Ellard, 4,224,446 to Catalucci and 3,989,703 to Niculescu-Duvaz et al. Synthesis of similar compounds are also disclosed in U.S. Pat. Nos. 4,077,957 to Piper et al and 2,742,468 to Brockman et al.
Of particular interest in this field is U.S. Pat. No. 4,374,987 to Singh et al which discloses a process for preparing highly pure methotrexate.
Methotrexate is widely used in cancer chemotherapy as an antifolate. The chemical is toxic to normal cells however, as well as to malignant cells. One hypothesis is that this general cytotoxicity is due to the formation of polyglutamate derivatives of the drug once it is inside the cell. Since these polyglutamate derivatives do not leave the cell as rapidly as the parent drug, the drug is in effect "trapped" in the cell. This results in the death of the cell and the observed general cytotoxicity.
Methotrexate (MTX) was first observed to produce temporary remission in leukemia in 1948. See an article by Farber etal in the New England Journal of Medicine, 238:787-792 (1948). As noted above, it has since been widely used to treat various neoplasms. The general mode of action for MTX is understood. MTX acts as a repressor of cellular growth by initiating a chain of events which ultimately leads to inhibition of thymidylate synthesis. Thymidylate synthesis is a crucial step in DNA biosynthesis. MTX acts by inhibiting dihydrofolate reductase (DHFR), an enzyme necessary for the reduction of dihydrofolate to tetrahydrofolate. Tetrahydrofolate is an essential cofactor which is able to acquire a methylene group from serine. Thymidylate synthase can then transfer this one carbon unit from N.sup.5, N.sup.10 -CH.sub.2 -tetrahydrofolate to 2'-deoxyuridine monophophate to generate thymidylate. In order for this reductive methylation to occur, functional DHFR must be coupled to a functional thymidylate synthase. This coupling is accomplished with the reduced forms of folic acid. MTX acts as a folate analogue which inhibits DHFR and uncouples it from thymidylate synthase. Rapidly proliferating cells, such as leukemias, are therefore most susceptable to MTX because of their accelerated DNA synthesis.
MTX has limited clinical value because of its cytotoxicity. When MTX is administered for leukemia chemotherapy, it is absorbed by normal host cells as well as the neoplasm. This causes inhibition of DHFR in normal as well as neoplastic cells. With high dosages of MTX, the normal host cells must be "rescued" by administering leucovorin. This is a derivative of tetrhydrofolate that can circumvent the inhibition of DHFR.
The cytotoxicity of MTX, is in turn believed to be related to the retention of polyglutamylated forms of MTX within the cells. It has been shown that polyglutamylated forms of MTX still capable of DHFR inhibition, leave the cells much less rapidly than unmetabolized (non-polyglutamylated) MTX.
Polyglutamate formation is a well established means by which cells modify folates in order to retain them. See an article in Archives of Biochemistry and Biophysics, Vol. 216, No. 2, July, pages 466-476, 1982, by Balinska et al.